The cloning of the genomes of hepatitis B virions of different serotypes is well known in the art (Miller et al). Dane particles which are hepatitis B virions and are isolatable from infected patients have a diameter of approximately 42 nm. Each consists of an envelope comprising the hepatitis B surface antigen (HBsAg), a capsid (HBcAg), an endogenous polymerase and a DNA genome. A third polypeptide, `e` antigen (HBeAg) is made by the hepatitis B virus and found in solubilized form in serum.
Infection with hepatitis B virus (HBV) is a serious, widespread problem but vaccines for use in mass immunisation are now widely available. Vaccines commercially available against HBV comprise hepatitis B virus surface antigen (HBsAg) either in native or recombinant form. The authentic (or wild type) hepatitis B virus surface antigen can be recovered from plasma of infected individuals as a particle of about 22 nm comprising of two proteins known as P24 and its glycosylated derivative GP28, both of which are encoded by a 226 amino acid coding sequence on the HBV genome known as the S protein coding sequence or HBV S-gene (Tiollais et al., (1985)). The complete amino acid sequence as well as the nucleotide sequence encoding, HBsAg is given in Valenzuela et al., Nature 280 815 (1979) (SEQ ID NO:1). The numbering system used by Tiollais et al. to define nucleotide and amino acid positions is used herein.
Insertion of HBV S-gene coding sequences under the control of yeast promoters on expression vectors to enable expression of HBsAg in S. cerevisiae for vaccine production has been described by Harford et al. in Develop. Biol. Standard 54 125 (1983), Valenzuela et al., Nature 298 347 (1982) and Bitter et al., J. Med. Virol. 25 123 (1988). Expression in Pichia pastoris has been described by Gregg et al., Biotechnology 5 479 (1987), (see also European Patent Publication No. 0226846) as has expression in Hansenula polymorpha (European Publication No. 0299108).
Vaccines have also been prepared from hybrid immunogenic particles comprising HBsAg protein as described in European Patent Publication No. 0278940. Such immunogenic particles can contain, for example, all or parts of the HBsAg precursor protein encoded by the coding sequence which immediately precedes the HBV-S gene on the HBV genome, referred to herein as the Pre-S coding sequence. The Pre-S coding sequence normally codes for 163 amino acids (in the case of the ay HBV sub type) and comprises a Pre-S1 coding sequence and a Pre-S2 coding sequence. The latter codes for 55 amino acids and immediately precedes the S protein coding sequence (European Publication No. EP-A-0278940).
The surface antigen (the S antigen) open reading frame of HBV-DNA is divided into three regions, pre-S1, pre S-2 and S. It encodes three envelope proteins of HBV termed: large, middle and major proteins. The major protein, HBsAg, consists of 226 amino acids and is encoded by the S gene (Tiollais et al., (1981); Tiollais et al., (1987) and Lau et al.). All three envelope proteins contain HBsAg antigenic sites and are easily detected by conventional immunoassays for HBsAg. These immunoassays are extensively used for diagnosing HBV infection and screening blood donors world-wide. The HBsAg reactivity is dependent on the structural conformation of the hydrophilic region from amino acids 124-147 which is defined as the `a` determinant (Ashton-Rickardt et al. and Brown et al.). This is common to all HBV subtypes and antibody to it confers protection against reinfection with any of the subtypes.
HBV-DNA sequences hybridising under highly stringent conditions with an HBV probe have been shown in the liver, serum and blood mononuclear cells of subjects negative for serum HBsAg (Brechot et al. (1985) and Thier et al). Recent results from different laboratories using dot blot hybridisation or Polymerase Chain Reaction (PCR) confirm the presence of HBV DNA sequences in serum from HBsAg negative subjects. The development of PCR techniques has permitted the detection of very low levels of HBV replication in many patients and has allowed sequencing of many isolates which have identified genetic variation in some isolates of the virus (Carman et al., (1990); Brechot et al., (1991); Blum et al.). In Taiwan and Sardinia, where HBV is highly endemic, 1.7% and 0.3% of HBsAg negative healthy blood donors had HBV DNA in their sera detectable by dot blot hybridisation (Lai et al., (1989) and Sun et al.) In mainland China, a molecular epidemiological investigation using PCR in anti-HB-positive individuals indicated the existence of HBV carriers with undetectable HBsAg, which accounts for 3% of the Chinese general population (Luo et al.)
Antigenic subtypes of HBV are defined serologically and have been shown to be caused by a single base changes in the region of the genome encoding HBsAg (Okamoto et al.). However, all presently known antigenic subtypes contain the `a` determinant consisting of amino acids 124 to 147 of HBsAg. Antibodies to the `a` determinant confers protection against all subtypes. It has been shown by in vitro mutagenesis that the cysteine at position 147 and the proline at position 142 are important for the exhibition of full antigenicity of the `a` determinant (Ashton-Rickardt et al.).
Additionally, Howard Thomas and William Carman, detailed in WO 91/14703 a variant of an HBsAg fragment in a vaccinated child born to an HBV infected mother. Sequencing revealed a point mutation from guanosine to adenosine at nucleotide position 587 resulting in an amino acid change from glycine to arginine at position 145 in the `a` determinant of HBsAg (see also Carman et al., (1990)). Similar HBV mutants have been reported to replicate in the host under humoral immune pressure, either actively (vaccine) or passively (hyperimmune globulin) induced (Okamoto et al., (1992) and McMahon et al.) However, the persistent presence of both HBsAg and anti-HBs (vaccine induced) detected by conventional immunoassays, in the serum of some of these patients, suggests that the mutation does not result in complete loss of antigenicity (Carman et al., (1990), Okamoto et al., (1992) and McMahon et al.).
From the clinical and epidemiological view, it is more important to investigate the molecular features of HBV from subjects without any HBsAg reacting in standard assays. Sequencing results from a Japanese patient who was HBeAg and HBV-DNA, as well as anti-HBs positive showed that amino acids 9 to 22 of the pre-S2 region were deleted whereas amino acids at position 3 and 8 of the pre-S2, and 126, 131 and 133 of the first loop of the `a` determinant of HBs were substituted (Moriyama et al.). Analysis of the deduced amino acid sequence from another isolate revealed substitutions at positions 3, 53 and 210 of the major HBs protein, all of which are outside the common `a` determinant and cannot really explain the absence of HBsAg in serum (Liang et al.).
During the last decade, several putative variants or mutants of hepatitis B virus have been described. For instance, McMahon et al. have described a substitution of arginine for glycine in a putative monoclonal antibody binding domain of HBsAg (as deduced by DNA sequence analysis) in a liver transplant patient treated with anti-HBsAg monoclonal antibody (Cold Spring Harbor Symposium on the Molecular Biology of Hepatitis B Viruses, September, 1989).
In an investigation by Carman et al. and the subject of patent application WO 94/26904, a mutant hepatitis B virus having a modified `a` determinant wherein two specific amino acids, asparagine and threonine, were inserted at position 122 of the HBsAg sequence. Further, a mutation at position 145, substituting glycine for the wild type arginine was also detailed in the amino acid sequence listing.
In another report, children and adults were found with circulating hepatitis B surface antigen, indicating viral replication, despite the presence of specific antibody (anti-HBs) after immunisation with one of two licensed hepatitis B vaccines (Zanetti et al.). Analysis of the HBsAg with monoclonal antibodies revealed that the circulating antigen did not carry the `a` determinant or that this determinant was masked. It was concluded that emergence of a variant of hepatitis B virus had been detected, possibly due to epidemiological pressure associated with immunisation in an endemic area of infection. The variant was, however, not characterised further.
From the work of Zanetti et al. it is clear that a great disadvantage with presently available hepatitis B vaccines is that they may, at least in a host with a predisposing immunogenetic make-up, cause the appearance of an `escape mutant`, i.e. a replicating infectious virus that has mutated away from neutralising immunity. Such a variant virus obviously has the capacity to cause disease and may be assumed to be transmissible. The variant virus may therefore give rise to a serious immunisation problem since it is not effectively neutralised by antibodies produced by vaccines based on normal HBsAg. Other mutations have been described in HBV, but their significance in terms of altered antigenicity is unclear (Moriyama et al. and Lai et al., (1990)).